Name: growing magnetotactic bacteria of the genus magnetospirillum: strains msr-1, amb-1 and ms-1
Uploaded: 2018-10-17T15:00:00
Description: Watch this Scientific Journal Video about Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1 at JoVE.com

We thank Richard B. Frankel for his help with MTB cultures, Adam P. Hitchcock and Xiaohui Zhu for their support while setting up the MTB cultures at McMaster University, and Marcia Reid for training and access to the electron microscopy facility (McMaster University, Faculty of Health Sciences). This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the US National Science Foundation.

1. Installation of the N2 Station
NOTE: Choose the inner diameter of the tubing so that it can be connected to the gas tank with minimum leakage and so that the cylinder of a 1 mL plastic syringe tightly fits in this tubing. An illustration of the complete N2 gassing station is provided in Figure 1.
<ol>
	<li>Safely install a N2 gas tank close to a bench on which there is enough space to set up the N2 station (a length...

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+magneto-aerotactic+behaviors+and+oxygen+sensing+mechanisms+in+cultured+magnetotactic+bacteria.">Diversity of magneto-aerotactic behaviors and oxygen sensing mechanisms in cultured magnetotactic bacteria.</a> Biophysical Journal. 107 (2), 527-538 (2014).</li><li>Heyen, U., Sch&#252;ler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+magnetosome+formation+by+microaerophilic+Magnetospirillum+strains+in+an+oxygen-controlled+fermentor.">Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor.</a> Applied Microbiology and Biotechnology. 61 (5-6), 536-544 (2003).</li><li>Blakemore, R. P., Maratea, D., Wolfe, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+pure+culture+of+a+freshwater+magnetic+spirillum+in+chemically+defined+medium.">Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium.</a> Journal of bacteriology. 140 (2), 720-729 (1979).</li><li>Oestreicher, Z., Lower, S. K., Lin, W., Lower, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+isolation+and+enrichment+of+naturally+occurring+magnetotactic+bacteria+from+the+environment.">Collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment.</a> Journal of Visualized Experiments. (69), (2012).</li><li>P&#243;sfai, M., Lef&#232;vre, M., Trubitsyn, C., Bazylinski, D. 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K., Pfennig, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+'capillary+racetrack'+method+for+isolation+of+magnetotactic+bacteria.">A 'capillary racetrack' method for isolation of magnetotactic bacteria.</a> FEMS Microbiology Ecology. 3 (1), 31-35 (1987).</li><li>Sch&#252;bbe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+spontaneous+nonmagnetic+mutant+of+Magnetospirillum+gryphiswaldense+reveals+a+large+deletion+comprising+a+putative+magnetosome+island.">Characterization of a spontaneous nonmagnetic mutant of Magnetospirillum gryphiswaldense reveals a large deletion comprising a putative magnetosome island.</a> Journal of Bacteriology. 185 (19), 5779-5790 (2003).</li><li>Nadkarni, R., Barkley, S., Fradin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+methods+to+measure+the+magnetic+moment+of+magnetotactic+bacteria+through+analysis+of+their+trajectories+in+external+magnetic+fields.">A comparison of methods to measure the magnetic moment of magnetotactic bacteria through analysis of their trajectories in external magnetic fields.</a> PloS One. 8 (12), e82064 (2013).</li><li>Waisbord, N., Lef&#232;vre, C. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosome+vesicles+are+present+before+magnetite+formation,+and+MamA+is+required+for+their+activation.">Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (11), 3839-3844 (2004).</li><li>Scheffel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+acidic+protein+aligns+magnetosomes+along+a+filamentous+structure+in+magnetotactic+bacteria.">An acidic protein aligns magnetosomes along a filamentous structure in magnetotactic bacteria.</a> Nature. 440 (7080), 110 (2006).</li><li>Zhu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+spectroscopy+and+magnetism+of+extracted+and+intracellular+magnetosomes+using+soft+X-ray+ptychography.">Measuring spectroscopy and magnetism of extracted and intracellular magnetosomes using soft X-ray ptychography.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (51), E8219-E8227 (2016).</li><li>Sch&#252;ler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+a+subcellular+compartment:+the+magnetosome+membrane+in+Magnetospirillum+gryphiswaldense.">Molecular analysis of a subcellular compartment: the magnetosome membrane in Magnetospirillum gryphiswaldense.</a> Archives of Microbiology. 181 (1), 1-7 (2004).</li><li>Kolinko, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+magnetic+nanostructures+in+a+foreign+organism+by+transfer+of+bacterial+magnetosome+gene+clusters.">Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.</a> Nature Nanotechnology. 9 (3), 193 (2014).</li><li>Hergt, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+properties+of+bacterial+magnetosomes+as+potential+diagnostic+and+therapeutic+tools.">Magnetic properties of bacterial magnetosomes as potential diagnostic and therapeutic tools.</a> Journal of Magnetism and Magnetic Materials. 293 (1), 80-86 (2005).</li><li>Lefevre, C. 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The specific O2 concentration requirements of MTB make them non-trivial to grow in the laboratory. A key step of the protocol for liquid medium is the initial removal of all O2 from the medium in order to control the final concentration by adding a definite volume of O2, just before inoculation. It has been shown that MSR-1 grows under almost fully aerobic conditions, however, the magnetism of the cells is drastically reduced. The results from the same study showed that strains AMB-1 and ...

Magnetotactic bacteria (MTB) represent a wide range of Gram-negative prokaryotes ubiquitous in freshwater and marine aquatic habitats1. These bacteria share the ability to produce magnetic crystals made of either magnetite (Fe3O4) or greigite (Fe3S4), which are in most cases assembled into chains inside the cells. This particular structural motif is due to the presence of several specific proteins acting both in the cytoplasm of the bacteria and on the lipid membrane that surrounds each crystal2. Each individual crystal and its surrounding membrane vesicle is called a magnetosome and is ranging in size from about 30 to 50 nm in Magnetospirillum species3. Because of the chain arrangement of magnetosomes, these bacteria possess a permanent magnetic dipole moment that makes them align passively with externally applied magnetic fields. Therefore, these bacteria actively swim along magnetic field lines, acting as self-propelled micro-compasses presumably to more effectively locate the most favorable conditions (e.g., O2 concentration) for growth.
An interesting property of MTB is their ability to regulate both the chemistry and the crystallography of their magnetosome crystals. Most strains produce relatively high purity crystals of either magnetite or greigite, although some biomineralize both minerals4. In all cases, the bacteria are able to precisely control the size and the shape of their single magnetic domain crystals. This explains why a great amount of research is undertaken to develop a better understanding of how MTB perform this biomineralization process. Understanding this process might allow the researchers to tailor-make magnetic nanocrystals for many commercial and medical applications.
A substantial obstacle to extensive research on MTB has been the difficulty of growing them in the laboratory. Most species, including the strains used in this work, are obligately microaerophilic when grown with O2 as a terminal electron acceptor. This explains why these bacteria are most often found at the transition zone between oxic and anoxic conditions (the oxic-anoxic interface, OAI). This clearly shows that MTB have precise O2 concentration requirements which obviously needs to be taken into account when devising growth media for these organisms. Moreover, the great existing diversity of MTB implies that different strains will need different types of chemical gradients and nutrients to achieve optimal growth.
In this work, we describe the methods for growing three of the most widely studied MTB: Magnetospirillum magneticum (strain AMB-1), M. magnetotacticum (MS-1) and M. gryphiswaldense (MSR-1). These species phylogenetically belong to the Alphaproteobacteria class in the Proteobacteria phylum, are helical in morphology and possess a polar flagellum at each end of the cell. We provide the protocols for growing strain MSR-1 in both liquid and O2 concentration gradient semi-solid media, based on previously published medium recipes5,6. We also present a detailed protocol for growing strains AMB-1 and MS-1 in modified Magnetic Spirillum Growth Medium (MGSM)7.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">AMB-1</td>
			<td style="width:437px;">American Type Culture Collection (ATCC)</td>
			<td style="width:119px;">ATCC 700264</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">MS-1</td>
			<td>ATCC</td>
			<td>ATCC 31632&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">MSR-1</td>
			<td>Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)</td>
			<td>DSM 6361</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Ferric citrate</td>
			<td>Sigma-Aldrich</td>
			<td>F3388-250G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Trace mineral supplement</td>
			<td>ATCC</td>
			<td>MD-TMS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">KH2PO4</td>
			<td>EMD</td>
			<td>PX1565-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">MgSO4.7 H2O</td>
			<td>EMD</td>
			<td>MX0070-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">HEPES</td>
			<td>BioShop Canada Inc</td>
			<td>HEP001.250</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">NaNO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5506-250G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Yeast extract</td>
			<td>Fischer scientific</td>
			<td>DF210929</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Peptone</td>
			<td>Fischer scientific</td>
			<td>DF0436-17-5</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Potassium L-lactate solution (60%)</td>
			<td>Sigma-Aldrich</td>
			<td>60389-250ML-F</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">D-(-)-Quinic acid</td>
			<td>Sigma-Aldrich</td>
			<td>138622</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">FeCl3.6H2O</td>
			<td>Fischer scientific</td>
			<td>I88-100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Vitamin supplement</td>
			<td>ATCC</td>
			<td>MD-VS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sodium succinate hexahydrate</td>
			<td>Fischer scientific</td>
			<td>S413-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sodium L-tartrate dibasic dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>228729-100G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sodium acetate trihydrate</td>
			<td>EMD</td>
			<td>SX0255-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Resazurin</td>
			<td>Difco</td>
			<td>0704-13</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544-25G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">K2HPO4</td>
			<td>Caledon</td>
			<td>6620-1-65</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">FeCl2 .4H2O</td>
			<td>Sigma-Aldrich</td>
			<td>44939-250G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sodium bicarbonate</td>
			<td>EMD</td>
			<td>SX0320-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">NaCl</td>
			<td>Caledon</td>
			<td>7560-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">NH4Cl</td>
			<td>EMD</td>
			<td>1011450500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">CaCl2.2 H2O</td>
			<td>EMD</td>
			<td>1023820500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Agar A</td>
			<td>Bio Basic Canada Inc</td>
			<td>FB0010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">L-cysteine.HCl.H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7880-100G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">1.0 mL syringes</td>
			<td>Fischer scientific</td>
			<td>B309659</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">25G&#160; x 1 needles</td>
			<td>BD</td>
			<td>305125</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">125 mL serum bottles</td>
			<td>Wheaton</td>
			<td>223748</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">20 mm aluminum seals</td>
			<td>Wheaton</td>
			<td>224223-01</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">20mm E-Z Crimper</td>
			<td>Wheaton</td>
			<td>W225303</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Butyl-rubber stoppers</td>
			<td>Bellco Glass, Inc.</td>
			<td>2048-11800</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Hungate tubes</td>
			<td>Chemglass (VWR)</td>
			<td>CLS-4208-01</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Septum stopper, 13mm, Hungate</td>
			<td>Bellco Glass, Inc.</td>
			<td>2047-11600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Glass culture Tubes</td>
			<td>Corning (VWR)</td>
			<td>9826-16X</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Hydrochloric acid 36.5-38%, BioReagent</td>
			<td>Sigma-Aldrich</td>
			<td>H1758-100ML</td>
			<td>11.6 - 12 N</td>
		</tr>
	</tbody>
</table>

growing magnetotactic bacteria of the genus magnetospirillum: strains msr-1, amb-1 and ms-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Successful preparation of the growth media can be assessed as follows. At the end of the process, clear solutions (i.e., free of any precipitate) should be obtained (this is true for both the liquid media and the O2 gradient semi-solid medium). A picture displaying the expected aspect of MSR-1 liquid medium before inoculation can be seen in Figure 2a. A successful O2 concentration gradient semi-solid medium is signaled by the fo...

Watch this Scientific Journal Video about Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1 at JoVE.com

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

This method allows growing three different species of magnetotactic bacteria called MSR-1, AMB-1, and MS-1. All freshwater species, and all from the genus magnetospirillum. It is an essential first step for any kind of reproducible research involving these organisms.Magnetotactic bacteria have very specific oxygen requirements, which is why they developed a magnetic navigation system. It is thus very important to control the amount of oxygen available to them. All of this protocol has been optimized for a few specific strains of freshwater magnetospirilla.Its details can be optimized in order to accommodate the oxygen and nutrient requirements of other magnetotactic bacteria. Safely install a nitrogen gas tank close to a bench on which there is enough space to set-up the nitrogen station. Connect a piece of tubing to the tank that is long enough to reach the area where the station will be built.If necessary, apply Teflon tape at the output of the tank to avoid any leakage. To build a station capable of bubbling five bottles of medium at the same time, cut four pieces of tubing of aproximately five centimeters in length. Assemble the pieces of tubing in a line with three 3-way T-shaped plastic fittings.Connect one end of this line to the piece of tubing at the output of the nitrogen tank through an extra T-shaped fitting. Add a 90-degree elbow fitting at the other end. Use tape to attach the structure to a horizontal metal rod placed aproximately 30 centimeters above the bench.Connect five pieces of tubing of aproximately 20 centimeters in length to the three outputs of the installed fittings. Remove the pistons from five one-milliliter plastic syringes, and cut the larger end of these syringes, keeping only the graduated part. Fill these syringes with cotton, taking care not to pack too tightly.Insert the syringes in the 20-centimeter long vertical pieces of tubing. Turn the gas on, and use soapy water to ensure that there is no leakage when nitrogen is flowing. Remove the caps of five 25 gauge needles and insert these needles into 10-centimeter pieces of thin tubing.Attach the prepared needles to the syringes of the nitrogen station. Ensure that nitrogen is flowing through all five lines, and keep the station on standby. Prepare a 10-millimolar ferric citrate solution by adding 0.245 grams of ferric citrate to 100 milliliters of distilled deionized water.Heat and stir to dissolve until an orange clear solution is obtained. Autoclave the solution using a standard cycle of at least 15 minutes exposure at 121 degrees Celsius before storing the sterilized stock solution at room temperature in the dark. To prepare five bottles of liquid growth medium for MSR-1, add the chemicals listed in a text protocol to a beaker containing 300 milliliters of distilled deionized water.Addition of the sterile ferric citrate and mineral solutions must be performed under the flame of a Bunsen burner using steril techniques. Sterile conditions are essential in order to avoid contamination of the culture by other bacterial species. After the addition of all chemicals, adjust the pH to 7.0 with one molar sodium hydroxide solution.Dispense the freshly prepared medium into 125-milliliter serum bottles, pouring 60 milliliters of medium in each bottle. Bubble nitrogen into the medium for 30 minutes to remove the dissolved oxygen using the small tubing connected to the nitrogen station. Place a butyl rubber stopper on top of each bottle, leaving a small opening to allow the excess gas to exit the bottle.After 30 minutes, crimp seal each bottle with the prepared stopper and an aluminum seal. The aluminum seal ensures that the bottle remains sealed during the rest of the protocol. Disconnect the needles and the thin tubing from the nitrogen station and replace them with clean needles.Adjust the valves of the nitrogen tank so that a gentle, continuous flow of gas exits the tank. Now, insert one of the needles connected to the nitrogen tank into a bottle of medium through the rubber stopper. Immediately insert another clean needle into the same bottle.Repeat this step for the other bottles, and let nitrogen flow for about 30 minutes to replace the air in the bottles by nitrogen. After 30 minutes, disconnect one bottle from the nitrogen station by removing the corresponding needle. Wait for a few seconds until the pressure in the bottle of medium decreases to atmospheric pressure, and remove the second needle.Repeat this step for all remaining bottles. To prepare 120 milliliters of semisolid growth medium for MSR-1, cover the beaker with aluminum foil and autoclave the solution prepared as described in the text protocol. Just before the end of the autoclave cycle, prepare a fresh 4%cysteine solution and adjust the pH to 7.0 with a five molar sodium hydroxide solution, as described in the text protocol.After autoclaving, let the medium cool down to 50 to 60 degrees Celsius, and bring the beaker under the flame of a Bunsen burner. Remove the aluminum foil and quickly add the chemicals listed in the text protocol using sterile techniques while gently stirring. Add 1.2 milliliters of filter sterilized cysteine solution.After the addition of all chemicals, transfer the warm medium into 16 milliliters sterile screw cap Hungate tubes. Transfer 12 milliliters of the medium into each tube, and seal the tubes. To inoculate strains MSR-1, AMB-1, and MS-1 in liquid medium, flame the tops of the oxygen and fresh medium bottles by applying ethanol on the stoppers and passing them through the flame of a Bunsen burner.Using a sterile syringe and a needle, extract one milliliter of oxygen from the oxygen bottle and transfer it into the fresh medium bottle. If using the inoculum from another culture grown in a glass bottle, flame both bottles. Then inoculate one milliliter of the older culture into the fresh medium.Incubate the culture at 32 degrees Celsius, and inoculate it into fresh medium after four to seven days. To inoculate MSR-1 in oxygen gradient, semisolid medium, verify that the tube of fresh medium displays a well-defined OAI, materialized by a pink to colorless interface about one to three centimeters below the surface of the medium. If the inoculum is coming from another oxygen concentration gradient semisolid culture, harvest the bacteria by aspirating 50 microliters of the culture with a sterile pipette tip placed on the band formed by the bacteria.Slowly inoculate these bacteria at the OAI in the fresh medium, avoiding disturbing the interface. Seal the tube and let the bacteria grow between 25 degrees Celsius and 30 degrees Celsius. To ensure that the bacteria are both magnetic and modal, place a drop of culture on microscope cover slip.Flip the cover slip, and install it on an O-ring resting on a glass slide. Ensure that the drop does not touch the bottom slide. Place the hanging drop under a microscope and focus on an edge of the droplet.Place the south pole of a magnet close to that edge, and watch the bacteria swim towards the edge. Flip the magnet to make them swim in the opposite direction. Shown here are the bottles of liquid media before inoculation and after bacterial growth.Turbidity in the second bottle indicates that bacteria are growing. This two times accelerated movie shows the expected result of a hanging drop experiment for a modal and magnetic magnetotactic bacteria culture. The bacteria initially swam towards the edge of the droplet when a magnetic field is applied.When the field direction of the magnet is reversed, the bacteria swam away from the edge where the magnetic field is applied. Shown here are tubes of semisolid medium before inoculation and after bacterial growth. As expected, a band of bacteria forms at the oxic-anoxic interface and migrates over time as oxygen is consumed and the interface moves up.Shown here is a representative electron micrograph of a magnetic spirillum. The flagella are indicated by the black arrows, while the red arrows show the magnetosomes. When attempting this procedure, it's very important to remember that magnetotactic bacteria have very specific oxygen level requirements in order to both grow properly and produce magnetosome.This protocol shows how to control and monitor the oxygen levels in the growth media in order to achieve the micro conditions preferred by these organisms. Following this method, high concentrations of healthy and highly magnetic cells can be obtained. This way, experiments that require large quantities of bacteria can be performed, such as magnetosome extraction, genomic and genetic studies, or observation of collective motion by microscopy.The possibility to grow magnetotactic bacteria in the lab is really what has allowed scientists exploring in a systematic way the fascinating biochemical and physical properties of these organisms.

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Research

JoVE Journal

Biology

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...